Antimicrobial and cytotoxic evaluation of some herbal essential oils in comparison with common antibiotics in bioassay condition.

BACKGROUND: Since ancient times, various infectious diseases have been treated using herbal drugs. Today, efforts regarding the discovery of the effectual components of plants possessing antimicrobial properties are advanced. Herbal essential oils are widely used for treatment of various diseases, and they play an important role in health care considerations.
METHODS: The antibacterial activity of Artemisia kermanensis, Lavandula officinalis, and Zataria multiflora Boiss essential oils against Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa (PTCC 1310), and Klebsiella pneumonia (PTCC 1053) was evaluated using the disk diffusion method as well as determination of the minimal inhibitory concentration and minimal bactericidal concentration. The composition of the three essential oils was determined with gas chromatography-mass spectrometry. Variable amounts of different components (such as oxygenated monoterpenes, thymol, carvacrol, and 1,8-cineol) were found in all three oils. Among the tested bacteria, S. aureus was the most sensitive to the three essential oils.
RESULTS: The obtained results showed that each of the three essential oils has an inhibitory effect on pathogenic strains. Of these three oils, Z. multiflora Boiss essential oil showed the highest inhibitory effect on microbial strains. Furthermore, comparison of the antibacterial effects of these three essential oils with ampicillin and tetracycline revealed that these antibiotics have a better effect in controlling pathogenic strains.
CONCLUSION: The essential oils used in the present study with different components showed antibacterial activity (especially Z. multiflora Boiss essential oil), and therefore they can be used as a new antibacterial substance.

Introduction

In recent decades, increasingly drug-resistant bacteria have been a major concern. Drug resistance is common among pathogenic staphylococci. Staphylococcus aureus is a facultative anaerobic Gram-positive coccobacillus naturally found in parts of the skin and nasal cavity. The inherent virulence of S. aureus and its ability to create a diverse array to life-threatening infections and capacity to adapt to different environmental conditions are the main concerns about this pathogen. Pseudomonas aeruginosa is Gram-negative aerobic motile basil.3, 4 This bacterium is commonly found in most environments in hospitals. P. aeruginosa often exist in small numbers in the normal intestinal flora and on human skin. It becomes pathogenic only when introduced into areas without normal defenses such as the skin and the mucus layer. This bacterium can express a variety of efficient efflux pump and antibiotic inactivating enzymes, so it can be resistant to antibiotics. Klebsiella pneumonia (which belongs to the Enterobacteriaceae family) is a nonmobile and encapsulated Gram-negative, facultative anaerobic bacillus, and is found in the normal flora of intestines. This bacterium causes infections in hospitals and communities. The majority of hospital infections caused by K. pneumonia are nosocomial pneumonia, urinary tract infections, diarrhea, and intra-abdominal infections. K. pneumonia may be attributable to multidrug efflux systems. The mounting concern about drug resistance has led researchers to focus more attention on natural products, including plants, with antimicrobial properties as the future source of antimicrobial agents.8, 9 For thousands of years, humans have been using natural products derived from plants for therapeutic purposes. The World Health Organization reported that in 2008, more than 80% of the world population depended on traditional medicine for their primary health care needs. Artemisia is a genus belonging to the Asteraceae family. Many members of this genus are important medicinal plants. Artemisia kermanensis is an endemic plant in Iran and important medicinal plant in the south of Kerman Province.11, 12 Lavandula officinalis (L. angustifolia) is an important species of the family Laminaceae, and is broadly distributed in the Mediterranean region. In Iranian flora, lavender is chiefly distributed in the northern parts of the country. Lavender oil is known for its excellent aroma and is widely used in flavor, perfume, and cosmetic industries; it is also recommended for its anti-inflammatory and anti-infectious effects. In Europe, lavender is used as an antispasmodic, carminative, and mild tranquilizer for digestive and mild nervous disorders. Lavender oil's antifungal and antibacterial activities oil have been reported.14, 15 Moreover, it has been found that lavender oil is active against many species of bacteria and fungi. For example, L. angustifolia oil was indicated to have in vitro antibacterial activity against methicillin-resistant S. aureus and vancomycin-resistant Enterococcus faecalis at a concentration of < 1%. Zataria multiflora Boiss, a member of the Labiatae family, is a native plant of Iran, Pakistan, and Afghanistan. It is traditionally used for anesthetic, antiseptic, and antispasmodic purposes. Z. multiflora has also been shown to have anti-inflammatory analgesic effects.17, 18 This plant is also used as a condiment and has many therapeutic applications in traditional folk medicine (Iranian Herbal Pharmacopoeia). In this study, we examined the antibacterial activity of A. kermanensis, L. officinalis, and Z. multiflora Boiss against three pathogenic bacteria (P. aeruginosa, S. aureus, and K. pneumonia).

Materials and methods

Origin and isolation of essential oils

Fresh Z. multiflora, L. officinalis, and A. kermanensis plants were gathered from Lorestan and Chaharmahal provinces in Iran (2012). Their scientific names were searched through the Herbarium part of Institution of Traditional Medicine in Iran (nos. 2359, 2360, and 2361, respectively). At first, the aerial parts of the herbs were kept at room temperature for 3 days, and after complete dryness was attained, the parts were powdered by mill. Making of essential oil was done with water using the essential making machine, Clevenger apparatus (model BP, Ashke Shisheh Co., Tehran, Iran & mantle model H610, Fater Electronic, Tehran, Iran) based on boiling point. For each batch, 100 g of the powder was placed in a 1-L balloon of Clevenger, and then water was added. After 5 hours of distillation, the essence—which was a yellow to green liquid with a good smell—was gathered. The oils were dried over anhydrous Na2SO4 and stored at 4 °C in sealed amber vials until use.

Gas chromatography-mass spectrometry

Analysis was carried out using a GC-mass chromatograph with an HP-5MS column (30 m × 0.25 mm, film thickness 0.25 m). Helium was used as the carrier gas at a flow rate of 0.8 mL/minute. The column temperature was kept at 50 °C for 2 minutes, and then it was programmed to 200 °C at a rate of 3 °C/minute and kept constant at 200 °C for 10 minutes. The injection was performed in split mode with ratio of 50:1 at 250 °C. The compounds were identified by comparison of the relative retention indices with those reported in the literature and also by comparison of their mass spectra with published mass spectra.20, 21 The retention indices for all the components were determined according to the Van Den Dool method using n-alkanes as standards.

Antimicrobial activities assays

Preparation of bacterial cells

The bacterial species consisted of S. aureus (ATCC 25923), P. aeruginosa (PTCC 1310), and K. pneumonia (PTCC 1053), which were prepared at the Traditional Medicine Institute of Isfahan (Isfahan, Iran). First, the Muller-Hinton agar (MHA) medium was prepared and transferred in sterilized Petri dishes (5 cm thick). Under aseptic conditions, the samples of bacteria were taken from basal culture using an applicator and then inoculated in the medium.

Antibacterial assay

In order to evaluate the antimicrobial effect, the disk diffusion method (which is known as Kirby–Bauer and is the most common form of antimicrobial assay) and assessment of minimal inhibitory and minimal bactericidal concentrations (MIC and MBC, respectively), were applied. After 18 hours of culture, liquid containing bacteria, with a standard density (1 × 106 CFU/mL) of 0.5 McFarland in MHA, was prepared, and by using Sampler, 500 μL of the liquid was transferred to MHA. The liquid was gently distributed on the surface of MHA using sterile loop. There were blank disks with 6 mm in diameter containing 30 μL with concentrations of 0.08 μg/disk, 0.16 μg/disk, 0.31 μg/disk, 0.63 μg/disk, 1.25 μg/disk, 2.5 μg/disk, 5 μg/disk, 10 μg/disk, 20 μg/disk, 40 μg/disk, 60 μg/disk, 80 μg/disk, and 100 μg/disk on MHA. A disk containing ampicillin, penicillin, and tetracycline was used as positive control, and the diameter of inhibition of zone was measured after 24 hours of incubation at 37 °C at 24 hours, 48 hours, and 72 hours in triplicate. The microwell method was used for determining the MIC value (the lowest concentration of an antibacterial agent that prevents visible bacterial growth after 24 hours of incubation at 37 °C) and MBC value (the lowest concentration required to kill certain bacteria) for S. aureus, P. aeruginosa, and K. pneumonia. The suspension of bacterial strain was prepared from the liquid culture with a standard darkness of 0.5 McFarland. The essential oils were prepared, and different dilutions (6 dilutions) were added to the pipes containing 10 mL liquid culture medium. In this step, in order to determine MIC, the 96-well plate was used. To every well, 95 μL Mueller–Hinton broth and 5 μL microbial suspension were added. Next, 100 μL of the essential oil with a concentration of 500 μg/μL was added to the first well. Then, 100 μL was taken from the first well and transferred to the next well. This process went on until the sixth well. The last well contained 195 μL MHB culture medium and 5 μL of microbial suspension without any essential oil) as negative control). In the next step, the ingredients of every well were mixed using a rotary shaker for 20 minutes. Then it was put in an incubator for 24 hours at a suitable temperature (37 °C). The microbial growth was measured at 600 nm.

In vitro analyses

Cell culture

The L929 fibroblast cell line of mouse (NCBI code 161 (was obtained from the Pasteur Institute of Iran (Tehran, Iran), then grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, 100 U/mL penicillin (Gibco), and 100 μg/mL streptomycin (Gibco, Carlsbad, CA, USA), at 37 °C in a humidified atmosphere containing 90% air and 5% CO2.

Cytotoxicity assay

In order to determine the cytotoxicity effect of essential oils, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used. This method is based on a mitochondrial succinate dehydrogenase activity that changes the yellow dye of MTT to the violet dye of Formosan. Formosan can dissolve in dimethyl sulfoxide, and its optical density (OD) could be measured using enzyme-linked immunosorbent assay.26, 27 The cells were cultured in T25, and after 90% confluency, they were removed from the culture dishes using trypsinization and suspended in 10 mL culture medium. Next, they were seeded with a cell count of 5 × 103 for L929 cells per well in 96-well plates for 24 hours. After that, the cultured cells were treated with different concentrations of essential oils (0 μg/mL, 0.75 μg/mL, 1.56 μg/mL, 3.12 μg/mL, 6.25 μg/mL, 12.5 μg/mL, 25 μg/mL, 50 μg/mL, 100 μg/mL, 150 μg/mL, 200 μg/mL, 250 μg/mL, 300 μg/mL, and 350 μg/mL), and the plate was incubated for 48 hours in a CO2 incubator at 37 °C. Next, 20 μL MTT solution was added to the wells and these were incubated for 4 hours. For dissolving Formosan christa, 100 μL dimethyl sulfoxide was added. The absorbance of MTT was measured at 560 nm. The vital percent of cells in negative control was considered 100, and it can be obtained using the following equation. A concentration from the essential oil that reduces cells’ vitality to half is observed as CC50.

Results

Data analysis was performed using SPSS version 20 (SPSS Inc., Chicago, IL, USA), analysis of variance (ANOVA), and Tukey's comparison procedure. After ANOVA showed significantly different values between treatment groups, Turkey's HSD (Honestly Significant Difference) test was performed for all pairwise comparisons which allows to rank means and put them into significant treatment groups, while controlling maximum experiment-wise error rate under null hypothesis. The effects of different concentrations of A. kermanensis, L. officinalis, and Z. multiflora Boiss essential oils were examined against the three bacteria (S. aureus, K. pneumonia, and P. aeruginosa) at 24 hours, 48 hours, and 72 hours using the disk diffusion method (Table 1, Table 2, Table 3). The results showed that the concentration of 100 μg/disk of each of the three essential oils was more efficient compared with lower concentrations on the bacteria (p < 0.0001).

Table 1

Antibacterial activity of different concentrations of Artemisia kermanensis essential oil against three bacteria using disk diffusion method (zone of inhibition)*

Artemisia kermanensis (μg/disk)	Staphylococcus aureusMean ± SE			Klebsiella pneumoniaMean ± SE			Pseudomonas aeruginosaMean ± SE
	24	48	72	24	48	72	24	48	72
0.08	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a
0.16	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a
0.31	0.50 ± 0.06a	0.63 ± 0.07a	0.63 ± 0.07a	0.17 ± 0.17a	0.27 ± 0.14a	0.27 ± 0.14a	0.10 ± 0.10a,b	0.17 ± 0.17a	0.17 ± 0.17a
0.63	2.49 ± 0.09b	2.67 ± 0.33b	2.67 ± 0.03b	1.20 ± 0.15b	1.37 ± 0.18b	1.50 ± 0.15b	1.33 ± 0.18b	1.60 ± 0.15b	1.60 ± 0.15b
1.25	4.20 ± 0.11c	4.33 ± 0.17c	4.40 ± 0.20c	2.27 ± 0.12c	2.50 ± 0.10c	2.63 ± 0.18c	3.33 ± 0.23c	3.63 ± 0.14c	3.63 ± 0.14c
2.5	6.48 ± 0.02d	6.51 ± 0.01d	6.52 ± 0.01d	4.00 ± 0.06d	4.13 ± 0.13d	4.37 ± 0.14d	5.13 ± 0.09d	5.37 ± 0.09d	5.37 ± 0.09d
5	7.90 ± 0.40e	8.02 ± 0.38d	8.11 ± 0.38e	6.57 ± 0.18e	6.73 ± 0.12e	6.90 ± 0.10e	7.57 ± 0.09e	7.73 ± 0.12e	7.73 ± 0.12e
10	9.64 ± 0.54f	10.02 ± 0.50e	11.16 ± 0.13f	8.27 ± 0.13f	8.70 ± 0.11f	8.70 ± 0.11f	8.53 ± 0.23e	9.13 ± 0.35f	9.13 ± 0.35f
20	12.13 ± 0.13g	13.30 ± 0.21f	13.40 ± 0.15g	13.20 ± 0.11g	13.80 ± 0.11g	13.80 ± 0.11g	10.80 ± 0.15f	11.10 ± 0.15g	11.33 ± 0.17g
40	14.43 ± 0.12h	14.70 ± 0.15f	14.87 ± 0.01g	15.17 ± 0.09h	15.77 ± 0.27h	15.97 ± 0.22h	12.20 ± 0.53g	12.40 ± 0.58h	12.40 ± 0.58g
60	16.70 ± 0.51i	17.23 ± 0.68g	17.50 ± 0.81h	17.13 ± 0.33i	17.57 ± 0.28i	17.57 ± 0.28i	13.83 ± 0.42h	14.20 ± 0.11i	14.20 ± 0.11h
80	19.77 ± 0.23j	20.10 ± 0.49h	20.17 ± 0.44i	19.97 ± 0.32j	20.23 ± 0.18j	20.40 ± 0.15j	15.87 ± 0.47i	16.10 ± 0.40j	16.10 ± 0.40i
100	22.27 ± 0.14k	22.73 ± 0.18i	22.73 ± 0.18j	25.70 ± 0.32k	26.37 ± 0.23k	26.37 ± 0.23k	17.43 ± 0.07j	17.67 ± 0.03k	17.67 ± 0.03j

Different letters on every column represent a meaningful difference (p < 0.0001).

SE, standard error.

Table 2

Antibacterial activity of different concentrations of Lavandula officinalis essential oil against three bacteria using disk diffusion method (zone of inhibition)*

Lavandula officinalis (μg/disk)	Staphylococcus aureusMean ± SE			Klebsiella pneumoniaMean ± SE			Pseudomonas aeruginosaMean ± SE
	24	48	72	24	48	72	24	48	72
0.08	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a
0.16	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a
0.31	0.37 ± 0.18a	0.40 ± 0.20a	0.40 ± 0.20a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.30 ± 0.15a	0.37 ± 0.18a,b	0.50 ± 0.25a,b
0.63	1.50 ± 0.06b	1.70 ± 0.06b	1.70 ± 0.06b	0.20 ± 0.20b	0.23 ± 0.23b	0.23 ± 0.23b	1.30 ± 0.21a,b	1.40 ± 0.15b,c	1.40 ± 0.15b
1.25	3.42 ± 0.21c	3.62 ± 0.21c	3.73 ± 0.09c	1.43 ± 0.23c	1.60 ± 0.21c	1.63 ± 0.23c	2.37 ± 0.18b,c	2.70 ± 0.06c,d	2.77 ± 0.03c
2.5	5.39 ± 0.05d	5.67 ± 0.12d	5.72 ± 0.16d	3.40 ± 0.25d	3.53 ± 0.24d	3.53 ± 0.24d	3.57 ± 0.14c	3.70 ± 0.11d	3.77 ± 0.14c
5	6.63 ± 0.20e	6.77 ± 0.18e	6.77 ± 0.18e	5.23 ± 0.14e	5.47 ± 0.09e	5.57 ± 0.09e	5.97 ± 0.03d	6.23 ± 0.12e	6.47 ± 0.14d
10	8.47 ± 0.12f	8.70 ± 0.12f	8.80 ± 0.15f	7.27 ± 0.14f	7.40 ± 0.11f	7.50 ± 0.11f	7.50 ± 0.11e	7.70 ± 0.06f	7.83 ± 0.03e
20	11.77 ± 0.19g	12.33 ± 0.18g	12.63 ± 0.18g	9.00 ± 0.25g	9.37 ± 0.28g	9.87 ± 0.13g	8.37 ± 0.18e	8.77 ± 0.18f	8.97 ± 0.18e
40	14.67 ± 0.20h	15.13 ± 0.09h	15.13 ± 0.09h	12.53 ± 0.09h	13.03 ± 0.09h	13.27 ± 0.14h	10.43 ± 0.14f	10.67 ± 0.09g	10.73 ± 0.14f
60	16.13 ± 0.07i	16.50 ± 0.06i	16.50 ± 0.06i	15.07 ± 0.18i	16.00 ± 0.40i	16.47 ± 0.34i	12.60 ± 0.17g	12.77 ± 0.14h	12.80 ± 0.17g
80	18.50 ± 0.06j	18.63 ± 0.09j	18.63 ± 0.09j	18.87 ± 0.09j	19.10 ± 0.15j	19.43 ± 0.22j	14.17 ± 0.12h	14.47 ± 0.14i	14.60 ± 0.10h
100	20.17 ± 0.09k	20.47 ± 0.09k	20.47 ± 0.09k	21.23 ± 0.56k	21.93 ± 0.54k	21.93 ± 0.54k	14.80 ± 0.81h	15.03 ± 0.84i	15.13 ± 0.75h

Different letters on every column represent meaningful difference (p < 0.0001).

SE, standard error.

Table 3

Antibacterial activity of different concentrations of Zataria multiflora Boiss essential oil against three bacteria using disk diffusion method (zone of inhibition)*

Zataria multiflora (μg/disk)	Staphylococcus aureusMean ± SE			Klebsiella pneumoniaMean ± SE			Pseudomonas aeruginosaMean ± SE
	24	48	72	24	48	72	24	48	72
0.08	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a
0.16	0.60 ± 0.23a	0.87 ± 0.23a	0.87 ± 0.23a	0.53 ± 0.14a,b	0.67 ± 0.14a	0.67 ± 0.14a	0.00 ± 0.00a	0.00 ± 0.00a	0.00 ± 0.00a
0.31	1.93 ± 0.29b	2.37 ± 0.24b	2.37 ± 0.24b	1.23 ± 0.12b,c	1.43 ± 0.27b	1.67 ± 0.24b	1.17 ± 0.17b	1.33 ± 0.17b	1.33 ± 0.17b
0.63	3.56 ± 0.29c	3.73 ± 0.18c	3.73 ± 0.18c	2.10 ± 0.10c	2.40 ± 0.15c	2.77 ± 0.14c	2.37 ± 0.12c	2.53 ± 0.09c	2.70 ± 0.06c
1.25	5.62 ± 0.06d	5.87 ± 0.09d	5.87 ± 0.09d	3.40 ± 0.00d	4.00 ± 0.00d	4.00 ± 0.00d	4.27 ± 0.18d	4.47 ± 0.22d	4.47 ± 0.22d
2.5	7.81 ± 0.13e	7.83 ± 0.12e	7.87 ± 0.13e	5.44 ± 0.02e	6.47 ± 0.03e	6.77 ± 0.03e	6.43 ± 0.12e	6.58 ± 0.09e	6.65 ± 0.05e
5	10.10 ± 0.45f	10.40 ± 0.45f	8.11 ± 0.38f	7.90 ± 0.40f	8.27 ± 0.37f	8.44 ± 0.34f	8.56 ± 0.23f	8.73 ± 0.12f	8.73 ± 0.12f
10	11.54 ± 0.12g	11.64 ± 0.07g	11.80 ± 0.06g	10.60 ± 0.24g	11.29 ± 0.05g	11.35 ± 0.05g	10.35 ± 0.26g	10.51 ± 0.13g	10.63 ± 0.12g
20	13.57 ± 0.12h	14.50 ± 0.26h	14.73 ± 0.13h	14.30 ± 0.26h	14.50 ± 0.29h	14.70 ± 0.26h	11.97 ± 0.20h	12.30 ± 0.15h	12.30 ± 0.15h
40	16.43 ± 0.09i	16.93 ± 0.12i	17.30 ± 0.15i	16.70 ± 0.11i	16.93 ± 0.07i	17.00 ± 0.11i	13.27 ± 0.13i	13.43 ± 0.18i	13.53 ± 0.22i
60	20.37 ± 0.20j	20.93 ± 0.07j	21.27 ± 0.14j	20.80 ± 0.30j	21.67 ± 0.09j	21.77 ± 0.09j	15.23 ± 0.14j	15.63 ± 0.14j	15.63 ± 0.14j
80	24.67 ± 0.03k	24.97 ± 0.03k	25.20 ± 0.15k	24.43 ± 0.12j	25.13 ± 0.13k	25.33 ± 0.20k	17.20 ± 0.11k	17.60 ± 0.30k	17.83 ± 0.23k
100	27.80 ± 0.20l	28.67 ± 0.33l	28.67 ± 0.33l	27.83 ± 0.12k	28.10 ± 0.21l	28.10 ± 0.21l	19.90 ± 0.27l	20.40 ± 0.23l	20.53 ± 0.18l

Different letters on every column represent meaningful difference (p < 0.0001).

SE, standard error.

In the highest concentration of A. kermanensis essential oil (100 μg/disk), most of the antibacterial effects was observed against K. pneumonia. However, at lower concentrations (from 80 μg/disk to 20 μg/disk), a similar effect was observed on both S. aureus and K. pneumonia. At concentrations < 20 μg/disk, a minimal antibacterial effect was observed against K. pneumonia. In a broad range of A. kermanensis essential oil concentrations used, P. aeruginosa (with the smallest inhibition zone compared with other bacteria) showed more resistance toward this oil (Fig. 1).

Fig. 1

Effect of essential oil of Artemisia kermanensis on three species of bacteria.

In concentrations < 20 μg/disk, Z. multiflora Boiss essential oil is more effective against S. aureus than against the two other bacteria (in these concentrations, the antibacterial effects against K. pneumonia and P. aeruginosa were rather similar). But with increasing concentrations (20–100 μg/disk), it had the same effect on both S. aureus and K. pneumonia. In a broad range of Z. multiflora Boiss essential oil concentrations used, P. aeruginosa, which has the smallest inhibition zone compared to other bacteria, showed more resistance toward this oil (Fig. 2).

Fig. 2

Effect of Zataria multiflora Boiss essential oil on three species of bacteria.

At high concentrations (80 μg/disk and 100 μg/disk), the essential oil of L. officinalis performed more effectively against K. pneumonia compared with the two other bacteria. However, at concentrations < 80 μg/disk, it was found to be more effective against S. aureus. In a board range of L. officinalis essential oil concentrations used, P. aeruginosa, which has the smallest inhibition zone compared to the other bacteria, proved to be more resistant toward this oil (Fig. 3).

Fig. 3

Effect of essential oil of Lavandula officinalis on three species of bacteria.

A comparison between the three plant essential oils (at a concentration of 100 μg/disk) and positive control antibiotics (ampicillin, penicillin, and tetracycline) demonstrated that Z. multiflora Boiss essential oil (at all time intervals) had a stronger antibacterial effect (bigger inhibition zone) against the three bacteria (Table 4).

Table 4

Comparison of different common essential oils concentrations and antibiotics effect on Staphylococcus aureus, Klebsiella pneumonia, and Pseudomonas aeruginosa

Treatment(μg/disk)	Staphylococcus aureusMean ± SE			Klebsiella pneumoniaMean ± SE			Pseudomonas aeruginosaMean ± SE
	24	48	72	24	48	72	24	48	72
Artemisia kermanensis (100)	22.27 ± 0.14c	22.73 ± 0.18d	22.73 ± 0.18d	25.70 ± 0.32d	26.37 ± 0.23d	26.37 ± 0.23d	17.43 ± 0.07c	17.67 ± 0.03c	17.67 ± 0.03c
Lavandula officinalis (100)	20.17 ± 0.09b	20.47 ± 0.09c	20.47 ± 0.09c	21.23 ± 0.56c	21.93 ± 0.54c	21.93 ± 0.54c	14.80 ± 0.81b	15.03 ± 0.84b	15.13 ± 0.75b
Zataria multiflora (100)	27.80 ± 0.20d	28.67 ± 0.33e	28.67 ± 0.33e	27.83 ± 0.12d	28.10 ± 0.21d	28.10 ± 0.21d	19.90 ± 0.27d	20.40 ± 0.23d	20.53 ± 0.18d
Ampicillin (10)	16.44 ± 0.29a	16.80 ± 0.25a,b	16.91 ± 0.31a,b	6.40 ± 0.61a	6.63 ± 0.59a	6.63 ± 0.59a	5.93 ± 0.35a	6.17 ± 0.43a	6.17 ± 0.43a
Penicillin (10)	14.93 ± 0.52a	14.93 ± 0.52a	14.93 ± 0.52a	5.87 ± 0.41a	6.00 ± 0.29a	6.00 ± 0.29a	6.33 ± 0.67a	6.50 ± 0.78a	6.50 ± 0.78a
Tetracycline (30)	19.47 ± 0.73b,c	19.63 ± 0.63c	19.63 ± 0.63c	13.30 ± 0.70b	13.40 ± 0.78b	13.40 ± 0.78b	6.00 ± 0.52a	6.40 ± 0.45a	6.40 ± 0.45a

* Different letters on every column represent meaningful difference (p < 0.0001).

SE, standard error.

The MIC and MBC results demonstrated that Z. multiflora essential oil, with lower MIC and MBC values than L. officinalis and A. kermanensis essential oils, showed a higher antibacterial activity against the three bacteria (Table 5, Table 6, Table 7). Compared with L. officinalis essential oil, A. kermanensis essential oil had a higher antibacterial activity (lower MIC and MBC values) against the three bacteria (Table 5, Table 6, Table 7).

Table 5

MIC and MBC of essential oils on Pseudomonas aeruginosa

No.	Extract	MIC (μg/mL)		MBC (μg/mL)
		MIC 50	MIC 90
1	Artemisia kermanensis	37	62	71
2	Zataria multiflora	31	58	63
3	Lavandula officinalis	41	75	92
4	Ampicillin	12	21	25
5	Penicillin	12	20	25
6	Tetracycline	6	11	15

MBC, minimal inhibitory concentration; MIC, minimal bactericidal concentration; SE, standard error.

Table 6

MIC and MBC of the essential oils on Klebsiella pneumonia

No.	Extract	MIC (μg/mL)		MBC (μg/mL)
		MIC 50	MIC 90
1	Artemisia kermanensis	30	54	68
2	Zataria multiflora	25	47	51
3	Lavandula officinalis	39	63	76
4	Ampicillin	10	17	24
5	Penicillin	13	19	26
6	Tetracycline	11	17	22

MBC, minimal inhibitory concentration; MIC, minimal bactericidal concentration.

Table 7

MIC and MBC of the essential oils on Staphylococcus aureus

No.	Extract	MIC (μg/mL)		MBC (μg/mL)
		MIC 50	MIC 90
1	Artemisia kermanensis	28	48	57
2	Zataria multiflora	18	35	43
3	Lavandula officinalis	32	52	69
4	Ampicillin	4	6	9
5	Penicillin	6	9	10
6	Tetracycline	5	7	10

MBC, minimal inhibitory concentration; MIC, minimal bactericidal concentration.

Cytotoxic effects on viability of L929 cells

Results from the cytotoxicity test showed that Z. multiflora and A. kermanensis essential oils have no cellular toxic effect up to 6.25 μg/mL, and L. officinalis oil showed no cellular toxic effect up to 12.5 μg/mL. With the increase in essential oil concentration, cellular resistance is considerably decreased. The CC50 for Z. multiflora, L. officinalis, and A. kermanensis is 123 μg/mL, 218 μg/mL, and 154 μg/mL, respectively (Fig. 4).

Fig. 4

Cytotoxic activity of different concentration of herbal medicine essential oils. Each bar represents the mean±S.D of three independent experiments.

Discussion

Nowadays, the resistance of bacteria is increasing against antibiotics. Consequently, research on exploration of new materials having antimicrobial properties is growing. As essences and herbal extracts have been historically used in treatment of diseases,28, 29 they can be good candidates in such studies. Herbal extracts possessing antimicrobial effects on a broad range of organisms, nutrient applicability, and fewer side effects (compared with common antibiotics), can be a replacement for antibiotics.30, 31, 32 Various studies have documented the antimicrobial activity of essential oils and plant extracts, such as A. kermanensis, L. officinalis, and Z. multiflora Boiss.33, 34, 35 In this study, we evaluated the antibacterial activity of A. kermanensis, L. officinalis, Z. multiflora Boiss essential oils against P. aeroginusa, S. aureus, and K. pneumonia. The results showed that the extract of Z. multiflora has the highest effect on all species of bacteria used. The highest effect was observed against S. aureus, with an MBC of 43 μg/mL (Table 7). Motevasel and coworkers reported that the high concentration of Zataria extract showed the best antimicrobial activities and killed numerous types of bacteria with no difference between pathogens and nonpathogens. In a research by Sharififar and coworkers, the effect of this extract was tested on K. pneumoniae and S. aureus, in which MIC was measured as 30 μg/mL and 21 μg/mL, respectively. In our study, the MIC90 of Z. multiflora against K. pneumonia and S. aureus was measured as 25 μg/mL and 18 μg/mL, respectively. Our results are compatible with those obtained by Sharififar et al. Owlia and coworkers tested the effect of the extract on P. aeruginosa, and their results showed an MIC of 64 μg/mL and an MBC of 128 μg/mL. In our study, the MIC and MBC of Z. multiflora essential oil on P. aeruginosa were measured as 31 μg/mL and 63 μg/mL, respectively. This indicates that the essential oil used in our study has a better effect. Moreover, in another study by El-Shouny and coworkers, the researchers determined the effect of Thymus vulgaris essential oil on P. aeruginosa, which is resistant to common antibiotics. The MIC was measured as 0.32 mg/mL (320 μg/mL). Another experiment reported the effect of T. vulgaris essential oil on S. aureus ATCC 25923, and yielded an MIC of 1.33 mg/mL.38, 40 Another experiment in 2008 reported the effect of Z. multiflora on K. pneumonia. The MIC was reported to be between 312 μg/mL and 624 μg/mL. Results of the study indicated that the essential oils possess a better effect on pathogens. The analysis of Z. multiflora Boiss essential oil with gas chromatography–mass spectrometry (GC-MS) showed 34 constituents representing 96.94% of the total oil. The major components consisted of thymol (33.05%), carvacrol (25.88%), and p-cymene (11.34%) (Table 8). Govaris and coworkers reported that carvacrol (80.15%) and thymol (4.82%) were the major components of Z. multiflora. Different studies indicated that essential oils containing thymol, carvacrol, or eugenol possess the highest antimicrobial properties. The antimicrobial effect of essential oil components such as thymol, menthol, and linalyl acetate may be caused by a perturbation of the lipid fractions of bacterial plasma membranes, which might be influenced by the membrane permeability and leakage of intracellular materials. Z. multiflora essential oil, with a high percentage of thymol and carvacrol, has a considerable antimicrobial activity. According to our results and previous research, thymol is the major compound of Z. multiflora oil. Sharif Roohani and coworkers (2007) (39.65%), Mahboubi and GhazianBidgoli (38.7%), and Saei-Dehkordi and coworkers (47.46%) reported thymol as the major component of Z. multiflora oil. The European Chemicals Agency reported that thymol has not shown chronic side effects or teratogenic effects in previous studies, and it can be considered a safe compound.35, 45

Table 8

Chemical composition of the essential oil of Artemisia kermanensis

No	Compositions	%	RI
1	Artemisiatriene	0.41	926
2	α-Pinene	0.54	934
3	Camphene	0.93	949
4	Verbenene	1.88	954
5	Benzaldehyde	0.11	960
6	β-Pinene	0.08	977
7	p-Menthatriene	0.57	993
8	Yomogi alcohol	2.67	1001
9	α-Terpinene	0.2	1016
10	p-Cymene	1.88	1024
11	1,8-Cineole	1.82	1030
12	Artemisia ketone	0.11	1032
13	trans-Carane	0.13	1050
14	gamma-Terpinene	‘	1056
15	Artemesia alcohol	1.48	1082
16	Styrene	0.82	1087
17	α-Thujone	13.83	1108
18	β-Thujone	6.23	1117
19	trans-Pinocarveol	1.39	1138
20	Camphen	4.13	1142
21	Camphore	10.23	1144
22	p-Menth-1,5-dien-8-ol	2.04	1147
23	1-Menthene	0.49	1156
24	Pinocarvone	1.37	1160
25	Borneol	1.97	1164
26	p-Mentha-1,5-dien-8-ol	4.38	1166
27	Terpinene-4-ol	1.01	1175
28	Naphthalene	0.73	1178
29	p-Cymen-3-ol	1.26	1182
30	α-Terpineol	0.72	1188
31	Verbenone	1.53	1206
32	Norbornane	0.36	1215
33	Cuminic aldehyde	1.1	1235
34	(+)-Carvone	0.48	1239
35	Carvotanacetone	0.28	1243
36	cis-Myrtanol	0.15	1247
37	Carvenone	0.12	1253
38	Chrysanthenyl acetate	1	1256
39	Cinnamic aldehyde-E	0.16	1264
40	Bornyl acetate	2.3	1280
41	Thymol	1.29	1286
42	Carvacrol	1.78	1297
43	α-Copaene	0.23	1368
44	Methyl cinnamate	0.15	1375.7
45	(Z)-Jasmone	0.22	1393.1
46	Methyleugenol	0.15	1399.3
47	trans-Caryophyllene	0.3	1395.6
48	α-Curcumen	0.15	1475.4
49	Spathulenol	0.25	1569
50	Caryophyllene	0.07	1644.5
Total	75.84

Orhan and coworkers observed the effect of L. officinalis essential oil against P. aeruginosa, K. pneumonia, and S. aureus, and reported an MIC of 32 μg/mL, 128 μg/mL, and 128 μg/mL, respectively. In our study, the MIC of L. officinalis essential oil on the bacteria was 41 μg/mL, 63 μg/mL, and 52 μg/mL, respectively. The comparison between our study and that of Orhan et al indicated that in our study L. officinalis essential oil was more effective in terms of its inhibitory effect on K. pneumonia and S. aureus. Changes in the antimicrobial properties of the essential oil in different concentrations can be attributable to the different amounts of flavonoid compositions or different active forms of flavonoids. According to GC-MS results, 69 constituents were identified in L. officinalis essential oil, representing 83.99% of the total oil and major components, and these included 1,8-cineol (12.01%), camphore (9.16%), verbenone (8.47%), alpha-pinene (7.58%), thymol (6.23%) (Table 9). Soković and coworkers reported that linalyl acetate (27.54%) and linalool (27.21%) are the most abundant components in L. angustifolia (L. officinalis) oil. Meanwhile, Hamad and coworkers reported that the major components of L. langustifolia oil are linalool (24.63%) and camphor (13.58%). 1,8-Cineole and camphor, which are used as useful substances in producing numerous drugs, have antiseptic properties.49, 50

Table 9

Chemical composition of the essential oil of Lavandula officinalis

No	Compositions	%	RI
1	α-Pinene	7.58	938
2	Camphene	4.51	952
3	Verbenene	0.64	956
4	1,3,5-Cycloheptatriene	0.03	972
5	β-Pinene	0.49	979
6	3-Octanone	2.19	988
7	β-Myrcene	1.18	993
8	3-Octanol	0.36	997
9	α-Phellandrene	0.05	1007
10	o-Isopropenyltoluene	0.08	1014
11	α-Terpinene	0.12	1017
12	p-Cymene	2.96	1025
13	1,8-Cineol	12.01	1033
14	gamma-Terpinene	0.08	1057
15	Linalool oxide	0.06	1072
16	Methyl banzoate	0.99	1088
17	Linalool	2.45	1100
18	Thujancis	0.81	1103
19	D-Fenchyl alcohol	0.28	1112
20	Pinocarveol	0.12	1138
21	Camphore	9.16	1144
22	Isopinocamphone	1.72	1158
23	Pinocarvone	0.13	1160
24	Pinocamphone	0.39	1171
25	Terpene-4-ol	1.27	1174
26	naphtalene	0.08	1177
27	p-Cymen-8-ol	0.23	1183
28	α-Terpineol	2.31	1188
29	Myrtenol	0.35	1194
30	No pol (terpene)	1.11	1203
31	Verbenone	8.47	1209
32	trans-Carveol	0.13	1215
33	β-Citronellol	0.1	1225
34	Pulegone	0.09	1235
35	Piperitone	0.04	1250
36	Cinnamaldehyde	0.05	1265
37	Borneol acetate	2.41	1281
38	Thymol	6.23	1288
39	2-Hydroxy-4-isopropyl-1-methylbenzene	0.17	1290
40	Carvacrol	4.14	1297
41	α-Terpinene	0.15	1329
42	Piperitenone	0.37	1335
43	α-Cubebene	0.06	1343
44	Thymyl acetate	0.06	1349
45	α-Copaene	0.43	1369
46	trans-Caryophyllene	0.47	1412
47	α-Humulene	0.22	1446
48	Farnesene	0.08	1451
49	β-Acoradiene	0.09	1460
50	gamma-Cadinene	0.16	1473
51	Zingiberene	0.1	1488
52	β-Himachalene	0.28	1492
53	delta-Cadinene	0.27	1516
54	α-Cedrene	0.15	1524
55	Germacrene B	0.06	1548
56	spathulenol	0.26	1567
57	Caryophyllene oxide	0.29	1572
58	α-Farnesene	0.06	1587
59	Butlidenephthalide	0.15	1642
60	3N Butylphthalide	4.62	1687
61	Butylidene dihydro-phthalide	0.09	1720
Total		83.99

Derakhshan and coworkers investigated the effect of Artemisia turcomanica, Artemisia khorassanica, Artemisia kopetdaghensis, and Artemisia ciniformis extracts against S. aureus ATCC 25923, and their results showed MIC to be 3 mg/mL, 2 mg/mL, 2 mg/mL, and 1.5 mg/mL, respectively. Another study reported on the effect of Artemisia absinthium extract on S. aureus, and reported an MIC of 52 μg/mL. We also investigated the effect of A. kermanensis extract on S. aureus. Our results yielded an MIC of 48 μg/mL (Table 7), which is compatible with that obtained by Blagojevic et al in 2006. Konatchiev and coworkers examined the effect of Artemisia distans extract on S. aureus. They reported an MIC of 20 μg/mL. A comparison between our results with those of Konatchiev et al shows that the A. distans extract possesses a better inhibitory effect on S. aureus compared with the A. kermanensis extract. Generally, in reference to different studies, it is revealed that the essential oil and extract of different species of Artemisia have an inhibitory effect against P. aeruginosa, K. kermanensis, and S. aureus. Generally, Gram-positive bacteria are more sensitive to herbal extracts when compared with Gram-negative ones. This can be attributed to their different cell wall structures. Gram-positive bacteria have mucopeptide compositions, whereas Gram-negative bacteria just have a thin layer of mucopeptide and most of their cell wall is made of lipoprotein and lipopolysaccharide. Hence, they are more resistant against antibacterial materials.54, 55 In A. kermanensis oil, 50 constituents were identified representing 75.84% of the total oil. The major components were alpha-thujone (13.83%), camphor (10.23%), and p-mentha-1,5-dien-8-ol (4.38%) (Table 10). Kazemi and coworkers determined the constituents of A. kermanensis oil using GC-flame ionization detection and GC-MS methods. They reported that the major components of this oil were isoborneol (21.5%) and camphor (9.8%). Sardashti and Pourramazani Harati also analyzed A. kermanensis oil with GC-MS, and reported the following results: in 1,8-cineole (56.55%), borneol (5.28%), and camphene (4.48%). Oxygenated monoterpenes (examples of this substance include linalool, α-terpineol, 1,8-cineole, borneol, camphor, and alpha, beta-thujone) are prevalent components of essential oils. Oxygenated monoterpenes have shown variable antibacterial activities. Based on season, geographical location, and the location of plants, the overall quality and quantity of the essential oil of species vary. Climate and soil conditions can also affect the composition of the oil, and differences in the major constituents of the different compounds of the essential oils can likely be attributable to differences in habitat conditions.57, 58

Table 10

Chemical composition of the essential oil of Zataria multiflora Boiss

No	Compositions	%	RI
1	α-Thujene	0.34	931
2	α-Pinene	3.88	937
3	Camphene	0.18	951
4	Verbenene	0.02	956
5	Sabinene	0.02	974
6	β-Pinene	0.68	979
7	β-Myrcene	0.68	993
8	α-Phellandrene	0.11	1007
9	Δ-3-Carene	0.04	1012
10	α-Terpinene	1.32	1016
11	p-Cymene	11.34	1025
12	Limonene	0.67	1032
13	1,8-Cineole	0.55	1030
14	gamma-Terpinene	4.73	1057
15	trans-Sabinene hydrate	0.27	1087
16	Linalool	1.46	1098
17	Borneol	0.37	1162
18	Terpinen-4-ol	0.82	1186
19	α-Terpineol	0.67	1191
20	Carvacrol methyl ether	0.77	1239
21	Carvol	0.77	1239
22	trans-Anethole	2.46	1281
23	Thymol	33.05	1285
24	Carvacrol	25.88	1297
25	Thymyl acetate	1.03	1311
26	Carvacryl acetate	0.69	1371
27	β-Caryophyllene	1.83	1412
28	Aromadendrene	0.84	1437
29	α-Humulene	0.09	1443
30	Germacrene-D	0.13	1473
31	Ledene	0.77	1491
32	cis-α-Bisabolene	0.09	1537
33	(+)Spathulenol	0.24	1579
34	Caryophyllene oxide	0.15	1589
Total		96.94

Conclusion

Essential oils possess a range of volatile molecules such as terpenes and terpenoids, and phenol-derived aromatic and aliphatic compounds, which might have bactericidal, virucidal, and fungicidal consequences. Essential oils directly affect the cell membrane of the pathogenic microorganism by causing an increase in permeability and leakage of vital intracellular elements, and finally disorder the cell respiration and microbial enzyme system.59, 60 In this study, three essential oils showed antibacterial effects against tested bacterial strains. Z. multiflora Boiss essential oil showed stronger antibacterial effects than the other essential oils. Z. multiflora oil has also been found to be active against clinical isolates of a broad spectrum of beta-lactamase-producing K. pneumoniae. It has been shown that Z. multiflora extract can inhibit the release of the deoxyribonuclease (DNase) enzyme and the making of enterotoxin in S. aureu. Due to the increasing problem of antibiotic resistance in bacteria, using these essential oils as natural and new antimicrobial substances can be useful.

Conflicts of interest

The authors declare no conflict of interest.